EP2104437B1 - Probiotic-containing food product and a protonated weak monoacid - Google Patents

Description

The present invention relates to a method for producing a fresh food product based on vegetable juices and / or milk, comprising a stable concentration of live probiotics which produce false tastes and / or gas in the initial matrix of the food product containing from 1 to 20 g / l of protonated lactic acid and having a pH of between 3 and 4.

The ingestion of live microorganisms called probiotics, including some strains of bacteria, especially those belonging to the genus Lactobacillus are particularly beneficial in terms of health. Indeed, they have been the subject of numerous studies demonstrating preventive clinical effects in various fields (for example in the areas of allergic manifestations, infectious diarrhea, inflammatory diseases) and on certain physiological functions (for example the digestion of lactose, intestinal transit, immunity). These probiotics are particularly capable of promoting a good functioning of the intestinal flora that is likely to interest the general population. Indeed, these bacteria produce, among others, bacteriocins and lactic acid which indirectly increase the digestibility of food, promote intestinal peristalsis, and accelerate the evacuation of stool. In addition, these bacteria produce certain vitamins of the B complex, and generally promote the absorption of vitamins and minerals, lower blood cholesterol, strengthen the immune system and lining the intestinal mucosa to protect against invasion and the activities of microorganisms. harmful.

As a result, agri-food industries have been trying for years to incorporate such bacteria into their products.

Such products with added bacteria are traditionally dairy products but other food products, especially those based on plants and in particular fruits, are interesting to develop for the agri-food industry market.

Fruit-based food products containing Lactobacillus bacteria are already known in the state of the art, for example in the international patent application. WO 00/70972 , and the European patent application EP 0113055 .

However, in fruit-based food products supplemented with lactobacilli, it has been possible to observe a growth and / or a bacterial activity which induces during the storage of products, an alteration of their qualities by a production of gas and false taste that makes them unfit for consumption.

Indeed, many microorganisms are, for example, capable of decarboxylating substituted cinnamic acids such as trans-4-hydroxy-3-methoxy-cinnamic acid (ferulic acid) and trans-4-hydroxy-acid. cinnamic (p-coumaric acid) which may be present in milk or vegetable juices to respectively form the following two volatile compounds: 3-methoxy-4-hydroxystyrene (4-vinyl guaiacol) and 4-hydroxystyrene (4-vinyl) phenol). These molecules are responsible for false-tastes like "phenolic, smoked, gloves, medicinal ...". The p-coumaric acid and ferulic acid decarboxylase activities were detected in bacteria of the genus Lactobacillus. In particular, the Lactobacilli known for these activities are as follows: L. brevis, L. crispatus, L. fermentum, L. plantarum, L. pentoses, L. paracasei ( Van Beek, S and Priest FG - 2000 - Decarboxylation of substituted cinnamic acids by lactic acid bacteria isolated during malt whiskey fermentation - Applied and Environmental Microbiology, 66 (12): 5322-8 ). Thus, via biotransformation pathways, strains of lactobacilli are able to generate false tastes from phenolic acids.

In addition, many strains of the genus Leuconostoc, Streptococcus and Lactobacillus are also capable of degrading malate, citrate, pyruvate, fumarate, tartrate and gluconate, which may be present in milk or vegetable juices to produce protein. gas ( Hegazi FZ, Abo-Elnaga IG, 1980. Degradation of organic acids by dairy lactic acid bacteria. Mikrobiologie der Landwirtschaft der Technologie und Umweltschutzes, 135 (3), 212 ). For example, organic acids such as malic acid or citric acid, when degraded, unless this assimilation is accompanied by excessive production of acetate which also generates false-tastes do not pose this problem of generating unpleasant tastes for the consumer. Nevertheless, the assimilation of these organic acids by bacterial strains will produce CO ₂ this time which will swell the packaging of the product. Indeed, these organic acids are naturally metabolized by certain species of lactobacilli to produce pyruvate (the central compound of metabolic cycles such as carbon metabolism) and CO ₂ ; moreover, the pyruvate is itself subject to decarboxylations increasing the amount of CO ₂ produced.

A food product based on fruit drinks or fruit puree and including stable live probiotics will have the advantage of providing the consumer with the benefits of fruits and probiotics.

The National Health Nutrition Plan recommends the minimum consumption of five servings of fruits and vegetables per day. Observations conducted by many scientists show that eating more fruits and vegetables helps reduce cholesterol levels, fat intake, and reduce the prevalence of childhood obesity.

A milk-based food product with stable living probiotics will have the added benefit of providing the consumer with the benefits of a dairy product and probiotics.

Several scientific studies suggest that probiotics can also play a leading role in health. Each probiotic strain can offer specific health benefits. These health benefits include: Improved functioning of the digestive system and strengthening of natural defenses. Some probiotics work by absorbing proteins and others produce vitamins. Some can also produce compounds that fight the spread of pathogenic bacteria and can play a role in the intestinal ecosystem.

It would be desirable for the food industry to be able to prepare such food products, without the problems of false tastes and gases, and this is the object of the present invention.

The inventors have discovered that by adding the initial matrix of the food product comprising probiotics which, naturally, produce false tastes and / or gas in said matrix, with lactic acid as weak monoacid, in a zone of pH defined, it prevented probiotics from producing false tastes and / or gas in the food product.

However, the inventors have also shown that only the protonated form of lactic acid, that is to say, in a medium whose pH is lower than the pKa of lactic acid as a weak monoacid, is effective in preventing probiotics from producing false tastes and / or gas in the food product.

These have been able to demonstrate that a pH of between 3 and 4, preferably between 3.4 and 4, more preferably between 3.4 and 3.7, was necessary to have an acceptable taste for the consumer, while allowing that a maximum of the lactic acid added as weak monoacids is in a protonated form. The present invention makes it possible in particular to provide products having a concentration greater than 50% in fruit and comprising live probiotics at a stable concentration.

However, since the weak monoacids are acids, the lactic acid will decrease the pH of an already acidic initial matrix (such as fruit juices) below these desired pH values (between 3 and 4). In addition, weak monoacids tend to have bad taste, it is not acceptable to put more than 50g / l of lactic acid in a food product. Therefore, the present invention relates to a method of manufacturing a fresh food product comprising a stable concentration of live probiotics that produce false tastes and / or gas in the initial matrix of the food product characterized in that it contains from 1 to 20 g / l of protonated lactic acid and in that it has a pH of between 3 and 4, which comprises inter alia the steps of adjusting the pH to a target pH of between 3 and 4 by addition of d an acid or a food base.

This fresh food product comprising a stable concentration of live probiotics which produce false tastes and / or gas in the initial matrix of the food product and characterized in that it contains from 1 to 20 g / l of protonated lactic acid, and in that it has a pH of between 3 and 4, is stored at a temperature of between 0 and 15 ° C., preferably between 4 and 10 ° C.

A fresh food product (that is to say preserved at a temperature between 0 and 15 ° C, preferably between 4 and 10 ° C) preferred in the sense of the present invention contains more than 2.2 g / l and up to at 20 g / l of protonated lactic acid, and its pH is between 3.4 and 4. Such a product thus has a protonated lactic acid content of greater than 2.2 g / l, with a maximum of 20 g / l, and a pH greater than 3.4, with a maximum value of 4.

Preferably, this amount of protonated lactic acid is that present in the product, at the end of its manufacture.

By fresh food product is meant according to the present invention a food product which will be stored between 0 and 15 ° C, preferably 4 and 10 ° C.

The term "fresh food product" is generally understood to mean products kept at 4 to 8 ° C., but it is known to one skilled in the art that the consumer's refrigerator is more generally at a temperature of between 4 and 10 ° C. This is why food products are formulated so that they can be stored at such temperatures (between 4 and 10 ° C).

By probiotic is meant live microorganisms that, when ingested in sufficient quantity, exert a positive effect on health beyond the traditional nutritional effects.

By living probiotics is meant, according to the present invention, probiotics whose survival rate, after 28 days, in a food product according to the present invention is greater than 60% and preferably greater than 80%.

The viability of probiotics is measured by counting techniques known to those skilled in the art such as mass count, surface count, Malassez cells, direct counting, turbidity, nephelometry, electronic counting, flow cytometry, fluorescence, impedancemetry, image analysis.

By stable concentration is meant, according to the present invention, a concentration of live probiotics that varies by at most 50%, preferably 10% over a period of 35 days from the addition of live probiotics to the initial matrix of food product, at a temperature of 10 ° C.

According to the present invention, the bacterial concentration in the food product is greater than 10 ⁵ , preferably greater than 10 ⁷ CFU / ml, even more preferably greater than 0.5 10 ⁸ CFU / ml. Most preferably, the concentration is from 0.5 to ⁸ and 1.5 to 10 ⁸ CFU / ml.

Thus, for example, at a starting concentration in the food product according to the present invention of 10 ⁸ CFU / ml, a stable concentration would be between 0.5 × 10 ⁸ CFU / ml and 1.5 × 10 ⁸ CFU / ml, preferably 0.9 × 10 ⁸ CFU / ml. ⁸ CFU / ml and 1.1 10 ⁸ CFU / ml.

In particular, these probiotics may be bacterial strains.

By bacterial strains, it is preferentially to designate, according to the present invention, lactic acid bacteria, of the genus Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., Leuconostoc spp. and in particular Lactobacillus casei, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus reuteri , Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium adolescents, Bifidobacteium infantis, Streptococcus thermophilus, Lactococcus lactis, or their mixture.

More particularly, the bacterial strains that are preferred according to the present invention are of the genus Lactobacillus, or Biflobacterium, preferentially Lactobacillus plantarum and even more preferentially the Lactobacillus plantarum strains deposited on March 16, 1995 under the number DSM 9843 at the Deutsche Sammlung von Mikroorganismen von Zellkuturen. GmbH or strains of Lactobacillus plantarum deposited on 4/04/02 under number CNCM I-2845 to the National Collection of Microorganism Cultures.

• elle répond aux critères probiotiques fixés par la communauté scientifique.
• elle est brevetée, caractérisée (RAPD, ribotypage) et sa classification est confirmée ;
• elle est GRAS (de l'anglais "Generally Recognized As Safe") ;
• elle est déjà présente à un taux de 10⁸ UFC/ml dans le produit ProViva® distribué par Skanemejerier et est consommée depuis 1994 ;
• elle présente une très bonne survie à pH acide inférieur à 4;
• elle est amylase négative donc ne dégrade pas la texture du produit fini.

The strain Lactobacillus Plantarum deposited on March 16, 1995 under the number DSM 9843 at the Deutsche Sammlung von Mikroorganismen von Zellkuturen GmbH is marketed by PROBI under the name Lactobacillus plantarum 299v®. This strain has many advantages for use as a probiotic in a fruit-based food product:

• it meets the probiotic criteria set by the scientific community.
• it is patented, characterized (RAPD, ribotyping) and its classification is confirmed;
• it is GRAS (Generally Recognized As Safe);
• it is already present at a rate of 10 ⁸ CFU / ml in the ProViva® product distributed by Skanemejerier and has been consumed since 1994;
• it has a very good survival at acid pH of less than 4;
• it is negative amylase so does not degrade the texture of the finished product.

• elle présente un fort potentiel de post acidification
• elle engendre des défauts organoleptiques importants liés à la synthèse d'acide acétique.
• elle dégrade l'acide citrique (e.g jus de citron, jus d'orange) ou l'acide malique (e.g jus de pomme ou de poire) avec production de gaz carbonique d'où des problèmes de gonflement possibles notamment si la chaîne du froid est rompue (c'est à dire dépassement de la température de 8°C).

However, this strain also has several disadvantages:

• it has a high potential for post acidification
• it gives rise to important organoleptic defects related to the synthesis of acetic acid.
• it breaks down citric acid (eg lemon juice, orange juice) or malic acid (eg apple or pear juice) with the production of carbon dioxide, which can cause swelling problems, especially if the cold chain is broken (ie exceeding the temperature of 8 ° C).

This strain therefore has many positive points but its use for the manufacture of food products does not achieve a quality (including organoleptic) acceptable because of this production of false tastes and / or gas.

It is the same for the strain of Lactobacillus plantarum filed on 4/04/02 under number CNCM I-2845 to the National Collection of Cultures of Microorganisms.

More particularly, the probiotics according to the present invention are probiotics that produce false tastes and / or gas in the initial matrix of the food product.

By initial matrix of the food product is meant, according to the present invention, a food product, preferably milk, a vegetable juice, preferably being a fruit juice or a vegetable juice or a fruit juice or vegetable reconstituted concentrate-based, probiotic-free, non-depleted in organic acids, but optionally including other substances such as, for example, sugar, water, flavorings, colorants, sweeteners, anti-oxygen agents , milk, preservatives, texture agents, proteins of animal origin (proteins of milk, whey ...) or vegetable (soy, rice ...) or extracts of plants (soy, rice .. .).

For example, if the food product made using the method according to the present invention is based on fruit juice, supplemented with live probiotics and low protonated monoacid, the initial matrix of the food product is a fruit juice.

Similarly, another example may be: if the food product made using the method according to the present invention is milk-based, supplemented with live probiotics and low protonated monoacid, the initial matrix of the food product is milk.

According to the present invention, the food product is preferably based on an initial matrix of vegetable juice and / or milk. This food product can be a fermented dairy product.

By "fermented dairy products" is meant more particularly fermented dairy products ready for human consumption, that is to say fermented dairy foods.

Here, the term "fermented milks" and "yoghurts" are particularly targeted. Said fermented dairy foods may alternatively be white cheeses or small Swiss.

The terms "fermented milks" and "yoghurts" are given their usual meanings in the field of the dairy industry, that is, products intended for human consumption, which are derived from acidifying lactic acid fermentation. a dairy substrate. These products may contain secondary ingredients such as fruits, vegetables, sugar, etc. For example, see French Decree No. 88-1203 of 30 December 1988 on fermented milks and yoghurt or yogurt, published in the Official Gazette of the French Republic of 31 December 1988.

Reference can also be made to the "Codex Alimentarius" (prepared by the Codex Alimentarius Commission under the auspices of FAO and WHO, and published by the FAO Information Division, available online at .codexalimentarius.net; cf more particularly Codex Alimentarius Volume 12 "Codex Standards for Milk and Milk Products", and "CODEX STAN A-1 1 (a) -1975").

The term "fermented milk" is thus reserved here to the dairy product prepared with a dairy substrate which has undergone a treatment at least equivalent to pasteurization, inoculated with microorganisms belonging to the species or species characteristic of each product. A "fermented milk" has not undergone any treatment to remove a constituent element of the dairy substrate used, and in particular has not undergone draining of the coagulum. The coagulation of "fermented milks" must not be obtained by other means than those resulting from the activity of the microorganisms used.

The term "yoghurt" is reserved for fermented milk obtained, according to local and constant use, by the development of specific thermophilic lactic bacteria called Lactobacillus bulgaricus and Streptococcus thermophilus, which must be found alive in the finished product, at the rate of at least 10 million bacteria per gram reported to the milk part.

In some countries, the regulation allows the addition of other lactic acid bacteria in yogurt production, including the additional use of Bifidobacterium and / or Lactobacillus acidophilus and / or Lactobacillus casei strains .

These additional lactic acid strains are intended to confer on the finished product various properties, such as the property of promoting the balance of the intestinal flora, or of modulating the immune system.

In practice, the term "fermented milk" is therefore generally used to designate fermented milks other than yoghurt, and may, depending on the country, be called "Kefir", "Kumiss", "Lassi", "Dahi", "Leben", "Filmjôlk", "Villi", "Acidophilus milk" for example.

The quantity of free lactic acid contained in the fermented milk substrate shall not be less than 0.6 g per 100 g when sold to the consumer and the protein content of the milk component shall not be less than that of a normal milk.

The name "fromage blanc" or "petit-suisse" is here reserved for unripened, unsalted cheese, which has been fermented by lactic acid bacteria only (no fermentation other than lactic fermentation).

The dry matter content of white cheeses may be reduced to 15 g or 10 g per 100 g of cottage cheese, depending on whether their fat content is 25% greater than 20 g, or not more than 20 g, for 100 g of white cheese, after complete desiccation. The dry matter content of a cottage cheese is between 13 and 20%. The dry matter content of a small Swiss cheese is not less than 23 g per 100 g of petit-suisse. It is generally between 25 and 30%. The Swiss and small Swiss cheeses are generally grouped under the name "fresh cheeses" conventionally used in the technical field of the present invention.

The term "vegetable juice" is intended to denote, according to the present invention, fruit juices or vegetable juices or reconstituted fruit or vegetable juices, based on concentrate, or juices extracted from soya, tonyu, rice, oats, quinoa, chestnut, almond or hazelnut.

The preferred fruits according to the present invention are: pear, strawberry, peach, pineapple, grape, apple, apricot, orange, banana, mango, cherry, cherry (sweet), plum, prune, blackberry, blueberry, raspberry, grapefruit, guava, kiwi, passion, papaya, lemon, quince, rose hips, lychee, pomegranate, or melon.

Preferably, the vegetable juice is fruit juice.

False tastes mean an abnormal taste for the food product. A false taste is unpleasant for the consumer so not sought. Thus, by way of example, for the food products manufactured using the method according to the present invention, the "earthy-hay" type of taste can be mentioned via the fermentation and the oxidation of the product, of the type " vinegar "or of the" phenolic, smoked, gloves, medicinal ... "type via the ferment from organic acids present in the product, and of" rancid "type via the presence of volatile fatty acids.

The "earthy-hay" type of taste is the consequence of the presence of compounds such as alpha terpineol, acetoine, inalcohol, 4-ethylphenol.

The "vinegar" type of taste is the consequence of the presence of acetic acid.

False taste like "phenolic, smoked, gloves, medicinal ..." is the consequence of the presence of compounds such as 3-methoxy-4-hydroxystyrene (4-vinyl guaiacol) and 4-hydroxystyrene (4- vinyl phenol).

The "rancid" taste is the consequence of the presence of compounds such as benzoic acid, 2-nonanone, decanoic acid, octanoic acid and dodecanoic acid.

For the purpose of the present invention, probiotics which produce false tastes are probiotics which, by their metabolism, will be responsible, directly or indirectly, for the presence of one or more of these compounds chosen from alpha terpineol, Acetoine, inalcohol, 4-phenol, acetic acid, 3-methoxy-4-hydroxystyrene (4-vinyl guaiacol) and 4-hydroxystyrene (4-vinyl phenol), benzoic acid, 2 -nonanone, decanoic acid, octanoic acid, dodecanoic acid.

"Positive" notes can also be detected in the product, such as, for example, "orange" or "fruity" notes. These tastes are not unpleasant for the consumer, they are not included in the "false taste" according to the present invention.

The content of molecules responsible for "false taste" is measured by solid phase microextraction (SPME) associated with a gas chromatograph (GC) coupled to a mass spectrometer (MS). This method has been developed specifically and has increased sensitivity while having good reproducibility and repeatability. SPME allows a specific concentration of target volatile molecules for better quantification and identification. GPC allows the separation of volatile molecules according to their polarity and their molar mass and thus obtain peaks corresponding to each molecule. The content of each molecule is expressed in peak area, ie in absorbance unit (AU) proportional to their concentration in the sample. Finally, the mass spectrometer allows on the one hand a certain identification of each molecule via their fragmentation in characteristic ions and on the other hand a second quantification of volatile molecules where the content is this time expressed in mass units.

1. a) sélectionner la matrice initiale d'intérêt pour le produit alimentaire (par exemple lait et/ou jus végétal)
2. b) ajouter à cette matrice initiale 10⁸ UFC/ml du probiotique à tester
3. c) conditionner le produit obtenu à l'étape b) (brique cartonnée type brique de lait, bouteille en plastique...)
4. d) Après 35 jours, mesurer la présence et/ou la teneur en molécules responsables de "faux-goûts" recherchés, par exemple le 3-methoxy-4-hydroxystyrene (4-vinyl guaiacol) et le 4-hydroxystyrene (4-vinyl phénol) (ou l'un des composés cités ci-dessus) par micro-extraction en phase solide (SPME) associée à un chromatographe en phase gazeuse (CPG) couplé à un spectromètre de masse (SM), tel que défini ci-dessus,
5. e) de manière optionnelle, faire tester le produit par un panel d'experts en analyse sensorielle qui attribuera une note entre 0 et 3, 0 étant la note pour « aucune présence de faux goût dans le produit testé » et 3 la note pour « présence de faux goût très forte », seuls les produits ayant une note comprise entre 0 et 1 étant acceptables organoleptiquement,
6. f) identifier les probiotiques qui produisent des faux goûts dans la matrice initiale du produit alimentaire comme ceux ajoutés dans les produits alimentaires pour lesquels on mesure à l'étape d) une présence en molécules responsables de "faux-goûts" recherchés, en particulier le 3-methoxy-4-hydroxystyrene (4-vinyl guaiacol) et le 4-hydroxystyrene (4-vinyl phénol) (ou l'un des composés cités ci-dessus) et/ou ceux ayant une note moyenne comprise entre 1 et 3 au test du panel d'experts en analyse sensorielle de l'étape e).

Thus a method for identifying a probiotic that produces false tastes in the initial matrix of the food product comprises the following steps:

1. a) select the initial matrix of interest for the food product (eg milk and / or vegetable juice)
2. b) add to this initial matrix 10 ⁸ CFU / ml of the probiotic to be tested
3. c) conditioning the product obtained in step b) (cardboard brick type milk brick, plastic bottle ...)
4. d) After 35 days, measure the presence and / or the content of molecules responsible for "false-tastes", for example 3-methoxy-4-hydroxystyrene (4-vinyl guaiacol) and 4-hydroxystyrene (4-vinyl) phenol) (or one of the compounds mentioned above) by solid phase microextraction (SPME) associated with a gas chromatograph (GC) coupled to a mass spectrometer (MS), as defined above ,
5. e) optionally, have the product tested by a panel of sensory analysis experts who will score between 0 and 3, with 0 being the rating for "no false taste in the product tested" and 3 the score for " presence of very strong false taste ", only products with a score between 0 and 1 being organoleptically acceptable,
6. f) to identify the probiotics that produce false tastes in the initial matrix of the food product, such as those added in food products for which step d) a presence in molecules responsible for "false-tastes" is sought, in particular the 3-methoxy-4-hydroxystyrene (4-vinyl guaiacol) and 4-hydroxystyrene (4-vinyl phenol) (or one of the compounds mentioned above) and / or those having an average score of between 1 and 3 at panel of experts in sensory analysis of step e).

By gas is preferably meant, according to the present invention, CO ₂ .

1. a) sélectionner la matrice initiale d'intérêt pour le produit alimentaire (par exemple lait et/ou jus végétal)
2. b) ajouter à cette matrice initiale 10⁸ UFC/ml du probiotique à tester
3. c) conditionner le produit obtenu à l'étape b) dans un conditionnement déformable (brique cartonnée type brique de lait, bouteille en plastique, pot de yaourt...)
4. d) Après 35 jours, mesurer la pression dans le conditionnement.
5. e) de manière optionnelle, faire tester le produit par un panel d'experts connaissant bien ce type de produit qui attribuera une note entre 0 et 5, 0 étant la note pour « aucune présence de déformation du conditionnement » et 5 la note pour « déformation maximale du conditionnement », seuls les produits ayant une note comprise entre 0 et 1 étant acceptables,
6. f) identifier les probiotiques qui produisent du gaz dans la matrice initiale du produit alimentaire comme ceux ajoutés dans les produits alimentaires pour lesquels on mesure à l'étape d) une pression dans le conditionnement et/ou ceux ayant une note comprise entre 1 et 5 au test du panel d'experts connaissant bien ce type de produit de l'étape e).

As well as to identify a probiotic that produces false tastes, to identify a probiotic that produces gas in the initial matrix of the food product, a method comprising the following steps is used:

1. a) select the initial matrix of interest for the food product (eg milk and / or vegetable juice)
2. b) add to this initial matrix 10 ⁸ CFU / ml of the probiotic to be tested
3. c) conditioning the product obtained in step b) in a deformable packaging (cardboard brick type milk brick, plastic bottle, yoghurt pot ...)
4. d) After 35 days, measure the pressure in the package.
5. e) optionally, have the product tested by a panel of experts familiar with this type of product, which will assign a score between 0 and 5, where 0 is the note for "no presence of package deformation" and 5 is the score for " maximum deformation of the packaging ', only products with a score between 0 and 1 being acceptable,
6. f) identifying the probiotics which produce gas in the initial matrix of the food product, such as those added in the food products for which a pressure in the conditioning and / or those having a score between 1 and 5 are measured in step d) the panel of experts knowing this type of product of step e).

• Après ensemencement de L. plantarum à forte concentration (10⁸ UFC/mL) dans un jus de fruits (jus d'orange par exemple), lors de la conservation du produit dans des contenants flexibles hermétiquement fermés par un opercule souple (ex : pot de yaourt, bouteille en carton,..), un dégagement gazeux peut être mis en évidence. Celui-ci peut par exemple être caractérisé par des mesures rhéologiques (mesure de la résistance de l'opercule à l'écrasement par une force longitudinale) ou des mesures de pression en piquant directement au travers de l'opercule avec une seringue couplée à un manomètre.

A pressure measuring method such as that used in step d) is described below:

• After inoculation of L. plantarum at high concentration (10 ⁸ CFU / mL) in a fruit juice (orange juice for example), when the product is stored in flexible containers tightly closed with a soft seal (ex: pot yoghurt, cardboard bottle, ..), a gas release can be highlighted. This can for example be characterized by rheological measurements (measuring the resistance of the operculum to crushing by a longitudinal force) or pressure measurements by pricking directly through the lid with a syringe coupled to a manometer.

For the purposes of the present invention, the term "flavorings" is intended to mean ingredients intended to give a flavor (that is to say a taste and / or an odor) to a foodstuff.

• soit ils renforcent la flaveur naturelle du produit alimentaire, ou la restituent partiellement si elle est trop faible (produits ayant perdu une partie de leur goût au cours du procédé de fabrication),
• soit ils remplacent un ingrédient apportant une note aromatique au produit fini (ex : yaourt arôme fraise).

Flavors are used for two main technological purposes:

• either they reinforce the natural flavor of the food product, or restore it partially if it is too weak (products having lost part of their taste during the manufacturing process),
• or they replace an ingredient bringing an aromatic note to the finished product (eg strawberry flavored yogurt).

According to the present invention, the preferred flavors are: apple, orange, red fruit, strawberry, peach, apricot, plum, raspberry, blackberry, currant, lemon, citrus, grapefruit, banana, pineapple, kiwi, pear, cherry, coconut , passion fruit, mango, fig, rhubarb, melon, multifruits, exotic fruits, vanilla, chocolate, coffee, cappuccino.

By dyes are meant substances capable of rendering the food product, strengthening or conferring a color.

According to the present invention, the preferred dyes are: beta carotene, carmine.

By sweetener, we mean substances capable of mimicking the sweetening power of sugar without providing the calories of sugar.

According to the present invention, the preferred sweeteners are: aspartame, acesulfame K, saccharin, sucralose and cyclamate.

By anti-oxygen agents is meant substances capable of avoiding or reducing the oxidation phenomena which cause, among other things, the rancidity of the fats or the browning of the cut fruits and vegetables.

According to the present invention, the preferred anti-oxygen agents are: vitamin E, rosemary extract.

By milk is meant milk of animal origin (eg cow, goat, ewe) or milks reconstituted from dairy ingredients.

Preservatives are substances intended to aid preservation by preventing the presence and development of undesirable microorganisms (for example, molds or bacteria responsible for food poisoning) in the final food product.

According to the present invention, the preferred preservatives are sorbic acid, ascorbic acid, and sulfur dioxide.

By texture agents is meant substances which improve the presentation or the behavior of the final food product. Texture agents may be emulsifiers, stabilizers, thickeners, or gelling agents. They can be used in the food product according to the present invention alone or in combination.

According to the present invention, the preferred texture agents are: pectin, carob seed, carrageenans, alginates, guar gum, xanthan gum, starch, mono and diglycerides of fatty acids.

By water is optionally meant osmosis water. The osmosis water limits the amount of minerals present in the final product, the minerals may also be responsible for false taste.

Potassium, chlorine, magnesium and calcium are indeed rather bitter in different forms (KCl, NH ₄ CL, CaCl ₂ , Ca acetate, LiCl, MgSO ₄ ...) while sodium, lithium and sulphates are rather salty and / or acidic depending on the form in which they are (salt form: NaCl, Na ₂ SO ₄ , Na tartrate, acid form: Na ₂ NO ₃ , Li acetate, salt form and acid: Na acetate , Na ascorbate, Na citrate). In addition to these direct effects on the sensory qualities of the products, these compounds can also have a "salting out" effect on the volatile molecules responsible for false tastes of "smoked, phenolic ..." type by favoring their passage in the vapor phase above of the product, thus increasing the perceived false-taste intensity.

In a preferred manner according to the invention, the food product comprises between 5 and 20 g / l of lactic acid as a low food monoacid, more preferably 10 to 20 g / l.

By low food monoacid is meant to designate according to the present invention, a weak monoacid capable of being consumed. This is lactic acid.

By protonated weak monoacid is meant according to the present invention a weak monoacid whose acid function has not given its proton H +.

A weak acid is an acid that does not dissociate completely in water: when a weak acid AH is brought into contact with water, the following reaction takes place: AH + H2O <=> A- + H3O +. The reaction is not total but balanced.

A weak acid, after giving up a proton H +, turns into a weak base.

A monoacid is an acid that includes only one acid function.

The weak monoacid in protonated form may be present naturally in the initial matrix of the food product, or added in the initial matrix of the food product by a method according to the present invention.

The advantage of using weak monoacids in protonated form is related to their antibacterial activity which depends on their ability to reduce the pH and their ability to release a proton. This latter capacity is a function of the pH of the medium as well as the pKa value of the acid in question, which is here lactic acid. When in protonated form, weak monoacids are fat soluble and thus have the ability to enter microbial cells. Once inside the cells, these weak monoacids are in a more alkaline medium; as a result, they release their proton and induce a decrease in intracellular pH. This modification influences the bacterial metabolism notably by inhibiting numerous enzymatic activities and forcing the bacterium to use its energy by expelling the protons. This phenomenon is the first reason for the decrease in bacterial activity. In addition, Russel (1992) also proposes an additional explanation based on the intracellular accumulation of anions consistent with the first phenomenon ( Russell, JB 1992. A review: another explanation for the toxicity of acid fermentation at low pH: anion accumulation versus uncoupling. J. Appl. Bacteriol. 73: 363-370 .)

The fresh food product made using the process according to the present invention preferably contains up to 7.5 g / l of lactic acid as a low food monoacid in any form (protonated or not). .

Preferably, the pH of the food product is between 3.4 and 3.7.

Preferably, the pH values mentioned according to the present invention are for a food product before packaging or at the end of its manufacture. Indeed, it can be observed in some cases, and for specific products such as yogurt acidification of the packaged food product during its conservation. This phenomenon is called post-acidification (specific acidification of product preservation).

Thus, for example a fresh food product at the end of its manufacture, and before conditioning having a pH of 4.5 can, after conditioning, and 30 days of storage (preferably between 4 and 10 ° C), have a pH of 3.9 due to post-acidification.

In particular, the fresh food product is composed of 80% water. Preferably, this food product may be a beverage, preferably based on fruit juice, reconstituted fruit juice based on concentrate and / or milk.

According to the present invention, fruit juices, orange juices and in particular NFC (Not From Concentrate) 10-12 ° Brix and reconstituted orange juice containing concentrate can be cited as FCOJ (Frozen Concentrate Orange Juice) at 66 ° Brix and other fruit juices concentrated between 10 and 70 ° Brix.

According to the present invention, the food product comprises between 20 and 99.99% of fruit juice, preferably between 50 and 99.99% of fruit juice.

According to the present invention, live probiotics included in the food product that produce false tastes and / or gas in the initial matrix of the food product are probiotics that degrade organic acids selected from the group consisting of: citric acid, tartaric acid, pyruvic acid, fumaric acid, and gluconic acid.

More particularly, these probiotics have the capacity to degrade these organic acids into CO ₂ and / or compounds generating false tastes.

Even more particularly, these organic acids are contained in the initial matrix of the food product.

By organic acids is meant, according to the present invention, including malic acid, citric acid, tartaric acid, pyruvic acid, fumaric acid, or gluconic acid.

A method of identifying strains that degrade organic acids may be as shown in Example 1.

1. a) ajout de 1 à 50 g/l d'acide lactique en tant que monoacide faible alimentaire dans la matrice initiale du produit alimentaires ;
2. b) mesure du pH du produit obtenu à l'étape a) ;
3. c) ajustement du pH du produit obtenu à l'étape b) à un pH cible compris entre 3 et 4, de préférence compris entre 3,4 et 4, par ajout d'un acide alimentaire plus fort que le monoacide faible alimentaire ajouté à l'étape a) si le pH mesuré à l'étape b) est supérieur au pH cible ou ajout d'une base alimentaire si le pH mesuré à l'étape b) est inférieur au pH cible : et
4. d) ajout de probiotiques vivants au produit obtenu à l'étape c),

de sorte que ledit produit alimentaire :

• contient de 1 à 20 g/l d'acide lactique protoné [HA] à l'issue de sa fabrication tel que calculé par l'équation suivante : $$\frac{\left[\mathrm{HA}\right]={\left[\mathrm{HA}\right]}_{\mathrm{Tot}}/\left(1+{10}^{\mathrm{pH}-3,86}\right)}{}$$
  

où [HA] représente la concentration de monoacide faible protoné alimentaire et [HA]_Tot représente la concentration de monoacide faible alimentaire, sous forme protonée et non protonée,
a un pH compris entre 3 et 4 et
est conservé à une température comprise entre 0 et 15°C, préférentiellement entre 4 et 10°C.The present invention therefore relates to a method of manufacturing a food product as described above, characterized in that it comprises the following steps:

1. a) addition of 1 to 50 g / l of lactic acid as a low-dietary monoacid in the initial matrix of the food product;
2. b) measuring the pH of the product obtained in step a);
3. c) adjusting the pH of the product obtained in step b) to a target pH of between 3 and 4, preferably between 3.4 and 4, by adding a stronger food acid than the low food monoacid added to step a) if the pH measured in step b) is greater than the target pH or addition of a food base if the pH measured in step b) is lower than the target pH: and
4. d) adding live probiotics to the product obtained in step c),

so that said food product:

• contains from 1 to 20 g / l protonated lactic acid [HA] at the end of its manufacture as calculated by the following equation: $$\frac{\left[\mathrm{HA}\right]={\left[\mathrm{HA}\right]}_{\mathrm{Early}}/\left(1+{10}^{\mathrm{pH}-3,86}\right)}{}$$
  

where [HA] represents the concentration of low protonated monoacid food and [HA] _Tot represents the concentration of low food monoacid, in protonated and non-protonated form,
has a pH between 3 and 4 and
is stored at a temperature between 0 and 15 ° C, preferably between 4 and 10 ° C.

According to the method of the present invention, in step d) between ⁵ × 10 ⁵ and 10 ⁹ CFU / ml of live probiotics are added, preferably between 0.5 × 10 ⁸ and 1.5 × 10 ⁸ CFU / ml of live probiotics, even more preferably 10 ⁸ CFU / ml.

In a preferred manner according to the invention, in step a) between 5 and 50 g / l of lactic acid are added as low food monoacids.

According to the present invention, the term "weak dietary monoacid" is intended to denote a weak monoacid capable of being consumed. This is lactic acid.

The food product made using the process according to the present invention comprises lactic acid in protonated form. The percentage of protonated form in a given amount of lactic acid as a weak monoacid depends on the pH of the product in which it is located.

où
[A-] est la concentration de la base et [HA] la concentration de la forme protonée de l'acide conjugué.In general, the concentration of lactic acid as weak monoacid in the protonated state is correlated with the pH value, according to the following formula: $$\mathrm{pH}=\mathrm{pK}+{\log }_{\mathrm{10}}\left[\mathrm{AT}-\right]/\left[\mathrm{HA}\right],$$

or
[A-] is the concentration of the base and [HA] the concentration of the protonated form of the conjugated acid.

These concentrations are easily measurable by a person skilled in the art by routine techniques (for example by HPLC).

• 1,1 g/L (13 mmol/L) d'acide lactique à pH 3
• 2,4 g/L (26 mmol/L) d'acide lactique à pH 4.

For example, to obtain 1 g / L of protonated lactic acid, it is necessary initially:

• 1.1 g / L (13 mmol / L) of lactic acid at pH 3
• 2.4 g / L (26 mmol / L) of lactic acid at pH 4.

Therefore, in the process according to the present invention, to obtain a food product comprising between 1 and 20 g / l, preferably more than 2.2 g / l and up to 20 g / l, protonated lactic acid it will be necessary to add to the initial matrix of the food product a quantity of lactic acid of greater than or equal to the amount of protonated lactic acid desired in the food product, ie between 1 and 50 g / l of lactic acid as weak monoacid.

• 23 g/L (253 mmol/L) d'acide lactique à pH 3
• 47 g/L (529 mmol/L) d'acide lactique à pH 4.

Indeed, for example, to obtain 20 g / L (222 mmol / L) of protonated lactic acid, it is necessary initially:

• 23 g / L (253 mmol / L) of lactic acid at pH 3
• 47 g / L (529 mmol / L) of lactic acid at pH 4.

By food acid is meant, according to the present invention, an acid capable of being consumed. According to the present invention, and preferably, the food acid is an organic acid, preferably an acid selected from orthophosphoric acid, citric acid, in particular citric acid monohydrate, ascorbic acid and acid. malic or their mixtures. More preferably, the food acid chosen is orthophosphoric acid. These acids are, of course, all "food grade".

By food base is meant according to the present invention a base suitable for consumption. According to the present invention, and preferably, the food base is chosen from NaOH, and salts, more particularly chosen from ammonium citrate, calcium citrate, sodium citrate, potassium salts, tricalcium dicitrate, and phosphate buffer.

According to the present invention, the target pH is preferably greater than 3.4 and up to 4, with still more preferred values between 3.4 and 3.7. A margin of error of pH with respect to the target pH of 0.1 is acceptable according to the present invention.

EXAMPLES EXAMPLE 1 Formation of Gases by L. plantarum strains DSM 9843 and L. plantarum I-2845 (deposited at the CNCM on 4/04/02) as a function of the fruit juice inoculated. I Materials and Methods: I.1. Preparation of bacterial suspensions and inoculation of fruit juices

A first preculture of 2 ml with strains DSM 9844 and I-2845 is carried out. This preculture is used to inoculate at 1% 100 ml of neutral SRM (ie 10 ⁸ -10 ⁹ cfu / ml). From this second preculture 3x 1000 ml in neutral MRS (ie 10 ⁸ -10 ⁹ cfu / ml) are inoculated.

• remplissage de 6 pots avec 330 mL de culture
• centrifugation de 10 min, 12 000 g, 20°C
• élimination du surnageant et addition de 165 mL de culture
• centrifugation de 10 min, 12 000 g, 20°C
• élimination du surnageant

For each strain, the centrifugations (Beckman JA-25, rotor JA-10) are carried out with the 500 ml pots as follows:

• filling 6 pots with 330 mL of culture
• 10 min centrifugation, 12,000 g, 20 ° C
• elimination of the supernatant and addition of 165 ml of culture
• 10 min centrifugation, 12,000 g, 20 ° C
• elimination of the supernatant

Each pellet obtained is then taken separately in the fruit juice to be tested and the suspension obtained is put back in the fruit juice brick which is carefully closed again afterwards.

I.2. Dosages of organic acids .

The technique used consists of separating the organic acids by high performance anion exchange chromatography (HPAEC). The detection of organic acids is carried out by suppressive conductimetry (SCD).

The chromatographic system used is a DIONEX brand (type DX600) comprising a suppressive conductimetric detection system. The thermostatic conductivity cell (type DS3) is coupled to an ASRS-ULTRA external self-suppression system (4mm). This electrolytic suppressor was used with a countercurrent Milli-Q water recirculation mode at a flow rate of 4 mL / min (approximately 15 psi pressure).

An AS11-HC type anion exchange column (4 mm) is associated with an AG11-HC type guard column. The elution rate is 1.5 mL / min.

II Results: II.1. Bacterial counts.

Bacterial counts are performed during product storage to evaluate the survival of L. plantarum in fruit juice matrices. <b> Table 1: </ b> Bacterial counts of <i> L. plantarum </ i> when storing fruit juice matrices at 10 ° C. Strain Time (j) Orange Apple Grape DSM 9843 D0 1.8.10 ⁹ cfu / mL 1.7.10 ⁹ cfu / mL 9.5.10 ⁸ cfu / mL J5 5.0.10 ⁹ cfu / mL 5.8.10 ⁸ cfu / mL 4.1.10 ⁹ cfu / mL 1-2845 D0 1.10 ⁹ cfu / mL 9.8.10 ⁸ cfu / mL 6.0.10 ⁶ cfu / mL J5 4.5.10 ⁹ cfu / mL 1.6.10 ⁹ cfu / mL 3.9.10 ⁹ cfu / mL

II.2. Demonstration of the consumption of organic acids during storage.

The organic acid assays were run at 0 and 5 days at the same time as the counts and the results are summarized in Table 1. <b> Table 2: </ b> Produced metabolites and organic acids consumed in the storage at 10 ° C of fruit juice containing <i> L. plantarum. </ i> Lots Swelling of the bottle pH Lactate product Acetate product Malate Consumed Citrate consumed mmol mmol mmol mmol Apple juice Witness D0 - 3.43 0.00 0.00 0.00 0.00 + DSM 9843 J5 ++ 3.38 27.93 4.44 6.72 0.21 + I-2845 J5 ++ 3.39 40.86 4.38 21,20 0.19 Orange juice Witness D0 - 3.34 0.00 0.00 0.00 0.00 + DSM 9843 J5 +++ 3.26 53.79 25.78 11.99 4.91 + I-2845 J5 ++ 3.27 48,90 15.60 13.26 1.42 Grape juice Witness D0 - 3.22 0.00 0.00 0.00 0.00 + DSM 9843 J5 ++ 3.23 40.79 10.38 23.50 2.09 + I-2845 J5 +++ 3.24 44.59 8.16 33.87 1.43

From the results presented in Table 2, it is clear that malic acid is the substrate most consumed by L. plantarum regardless of the strain considered. This consumption is accompanied not only by lactate and acetate production and therefore by a significant drop in pH (especially in orange and apple juice), but also by a gas production having a macroscopic effect. on the packaging.

According to the metabolic pathways presented in FIG. 1, the absence of detection of product formate (no action of pyruvate formate lyase), the very low content of pentoses of the processed fruit juices, the following CO ₂ production budget (expressed in moles) can be proposed: $${\mathrm{CO}}_{\mathrm{2}}\mathrm{total}=\mathrm{malate\; consumed}+\mathrm{consumed\; citrate}+\left(\mathrm{total\; acetate\; product}-\mathrm{acetate\; from\; citrate}\right)$$

Or, replacing the acetate produced from the citrate by the amount of citrate consumed: $${\mathrm{CO}}_{\mathrm{2}}\mathrm{total}=\mathrm{malate\; consumed}+\mathrm{total\; acetate\; product}$$

Conclusion:

Malic acid and, to a lesser extent, citric acid therefore play a major role in the production of gas when 10 ° C high-dose fruit juice (> 1.10 ⁹ cfu / mL) of L- bacteria is stored. plantarum DSM 9843 or I-2845.

EXAMPLE 2

Objective: to show that impacting only the pH does not reduce the activity of L. plantarum used in a fruit juice.

An orange juice diluted to 20% is inoculated to reach an initial population of 1.10 ⁸ cfu / ml. Various acids and mixtures of acids are added to be in a pH range between 3.3 and 3.4.

After conditioning in jars of yoghurt hermetically sealed by an "aluminum" operculum, the pots are kept at 10 ° C. and the monitoring of the swelling (the production of CO ₂ allowing a macroscopic visualization of the bacterial activity) is carried out. <b> Table 3: </ b> Type of acid percentage in the finished product for pH = 3.3 pH Low protonated monoacid content (g / l) visible swelling of products stored at + 10 ° C malic acid qs 3.37 / D + 7 citric acid qs 3.38 / D + 7 Lactic acid 0.40% 3.40 3.0 J + 30 * orthophosphoric acid qs 3.30 / D + 7 lactic acid / citric acid 0.30% / 0.15% 3.40 2.2 J + 8 lactic acid / citric acid 0.40% / 0.15% 3.30 3.1 J + 15 * lactic acid / sodium citrate 0.40% / 0.06% 3.30 3.1 no to D + 7 * Lactic acid 0.50% 3.30 3.9 no to D + 7 * *: organoleptic perception not acceptable to consumers.

When stored at 10 ° C, for the same pH (3.37-3.40), the swelling kinetics are faster if the pH is reached by adding malic and citric acids. On the contrary, the presence of lactic acid makes it possible to prevent this swelling. However, lowering the pH of the product to values close to 3.3 with lactic acid (ie 0.4-0.5%), in association or not with citric acid or sodium citrate , is not acceptable by the consumer because the negative organoleptic impact of lactic acid is very important (perception very acidic, pungent, ...).

The pH is not the leverage factor to reduce the metabolic activity of L. plantarum but lactic acid having a certain effect, the impact of the pH / acidity dissociation was tested in Example 3.

EXAMPLE 3

Objective : to define the couple lactic acid / pH content to reduce the metabolic activity while maintaining a large bacterial population (> 10 ⁸ cfu / mL).

Matrix: orange juice 100% pure juice (original pH = 3.6).

Add lactic acid and, if necessary, adjust the pH by adding sodium hydroxide to reach the target value.

• Populations bactériennes initiales : 2,5.10⁸ ufc/mL
• Populations bactériennes à J+21 :
  • Essai n°1 : 2.10⁶ ufc/mL
  • Essai n°6 : 6.10⁷ ufc/mL => très mauvaises qualités organoleptiques.
  • Essai n°8 : 4.10⁷ ufc/mL
  • Essai n°10 : 4,5.10⁷ ufc/mL

Seeding: 0.015% of L. plantarum strain DSM 9843 at 1.4 × 10 ¹¹ cfu / ml in 100 ml of orange juice. <b> Table 4: </ b> Test no. 1 2 3 4 * 5 * 6 7 8 9 10 pH 3.5 4.5 4 4 4 3.6 4.5 4 4.5 3.5 [lactic acid] (g / L) 10 0 0 5 5 0 5 10 10 5 Low protonated monoacid content (g / l) 7 0 0 2.1 2.1 0 0.9 4.2 1.8 3.5 *: repetitions of the same conditions.

• Initial bacterial populations: 2.5 × 10 ⁸ cfu / mL
• Bacterial populations at D + 21:
  • Test n ° 1: 2.10 ⁶ cfu / mL
  • Test No. 6: 6.10 ⁷ cfu / mL => very poor organoleptic qualities.
  • Test n ° 8: 4.10 ⁷ cfu / mL
  • Test No. 10: 4.5.10 ⁷ cfu / mL

Regardless of the initial pH, the products not containing lactic acid (Tests Nos. 2, 3 and 6) all swelled as of D + 7. On the same date, the high pH, low lactic acid test (Test No. 7) slightly inflated. No other tests inflated. This result confirms once again that pH alone has no influence on the metabolic activity of L. plantarum.

At D + 21, trials 1, 4, 5, 8, 9 and 10 did not inflate. For the test n ° 1, this absence of swelling is linked to the weak bacterial survival (decrease of a factor 100 of the bacterial population). On the other hand, in the case of the other tests, the absence of swelling is concomitant with good survival of the bacteria (population still greater than 4.10 ⁷ cfu / ml). This makes it possible to highlight the importance of the choice of the "pH / lactic acid concentration" pair.

From all these tests, a target pH / lactic acid content pair could be defined in order to reduce the metabolic activity of L. plantarum (cf Example 4). The dose of target lactic acid will be adjusted by addition of molar lactic acid and the pH will be adjusted by orthophosphoric acid molar. Indeed, the latter, usable in the food industry and effective to rapidly reduce the pH, has no effect on the decrease in metabolic activity (see Example 2).

EXAMPLE 4 : first application on a dairy product to be drunk with fruit.

• pH nominal du produit fini ou abaissé à 3,9 et 3,7 par ajout d'acide orthophosphorique,
• Acidité lactique nominale du produit fini ou augmentée à 5,0 g/l par ajout d'acide lactique.

The purpose of the trial is to evaluate the impact on the activity of L. plantarum (population and taste in the product) of the following conditions:

• nominal pH of the finished product or reduced to 3.9 and 3.7 by addition of orthophosphoric acid,
• Nominal lactic acidity of the finished product or increased to 5.0 g / l by addition of lactic acid.

1. Product formula:

Table 5: constituents Percentage by mass (%) Skimmed milk 56.91 Cream 40% fat 2.50 Skim milk powder 0.38 Cristalized sugar 5.20 Lactic ferments of yogurt 0.01 Fruit preparation 15,00 Sterile water + sterilized acid solutions 20.00 TOTAL 100.00

2. Preparation processes:

The mixture skim milk, cream, milk powder and sugar is made, pasteurized, and then fermented at 38 ° C by adding lactic ferments.

The fermentation is stopped at pH 4.60 by cooling to 4 ° C.

The white mass obtained is then supplemented with fruit preparations, acid solutions, sterile water and frozen L. plantarum according to the table presented in paragraph 3.

3. Details of the tests:

In the following table are detailed the characteristics of the different mixtures tested for 1 kilogram of finished product. <b> Table 6: </ b> Mix number M1 M2 M3 M4 M5 M6 Amount of lactic acid solution added (ml) 0 0 0 4.33 4.33 4.33 Amount of added molar phosphoric acid solution (g) 0 13.8 24.3 0 12.1 21.0 pH finished product 4.29 3.9 3.7 4.22 3.9 3.7 Lactic acidity of the finished product 4.7 4.7 4.7 5.0 5.0 5.0 Low protonated monoacid content (g / l) 1.3 2.2 2.8 1.6 2.4 3.0 Quantity of L. plantarum added (g) 1 1 1 1 1 1 Initial population in L. plantarum (cfu / g) 1.10 ⁸ 1.10 ⁸ 1.10 ⁸ 1.10 ⁸ 1.10 ⁸ 1.10 ⁸

4. Evolution and evaluation of products during their lifetime.

In Table 7 which follows is detailed the characteristics of the different mixtures tested during their aging at a storage temperature of 10 ° C. <b> Table 7: </ b> Mix number M1 M2 M3 M4 M5 M6 pH J0 4.29 3.9 3.7 4.22 3.9 3.7 pH D + 8 days 3.53 3.40 3.33 3.51 3.45 3.37 pH D + 28 days 3.56 3.50 3.47 3.55 3.53 3.50 pH D + 35 days 3.52 3.46 3.44 3.54 3.47 3.46 Population in initial L. plantarum (cfu / g) 1.10 ⁸ 1.10 ⁸ 1.10 ⁸ 1.10 ⁸ 1.10 ⁸ 1.10 ⁸ Population in L. plantarum at D + 21 days (cfu / g) 21.10 ⁸ 15.10 ⁸ 11.10 ⁸ 15.10 ⁸ 8.8.10 ⁸ 7.8.10 ⁸ Visual evaluation of the swelling of the pots at D + 21 days Rating of 0 = absence at 5 = max 5 4 3 3 2 1 Evaluation of the characteristic odor of L. plantarum activity at D + 21 Score of 0 = absence at 3 = max 3 2 1 2 1 0.5 Evaluation of the characteristic taste of L activity. plantarum at D + 21 Score of 0 = absence at 3 = max 3 2 1 2 1 0.5

5. Conclusions:

For the same lactic acid content, the bacterial growth of L. plantarum is reduced by a lowering of the pH.

For the same pH value, the bacterial growth of L. plantarum is decreased by an increase in the lactic acid content.

At least this bacterial growth is 0.8 log.

For all the conditions of pH and lactic acidity tested, the pH of the finished product at 35 days is between 3.44 and 3.54. Therefore, it may be interesting to place directly in this pH area from the manufacture of the product. This is what was tested in Example 5 with the comparison of the results obtained at pH 3.7 and 3.45.

EXAMPLE 5: Second application on a dairy product to be drunk with fruit.

• pH du produit fini abaissé à 3,7 et 3,45 par ajout d'acide orthophosphorique,
• Acidité lactique nominale (5,4g/l) du produit fini ou augmentée à 7,5 g/l par ajout d'acide lactique.

Following the test described in Example 4, a new test is carried out in order to evaluate the impact on the activity of L. plantarum (population and taste in the product) of the following modified conditions:

• pH of the finished product lowered to 3.7 and 3.45 by addition of orthophosphoric acid,
• Nominal lactic acidity (5.4 g / l) of the finished product or increased to 7.5 g / l by addition of lactic acid.

1. Product formula:

Table 8 constituents Percentage by mass (%) Skimmed milk 54.76 Cream 40% fat 2.49 Skim milk powder 0.59 Cristalized sugar 7.15 Lactic ferments of yogurt 0.01 Fruit preparation 15,00 Sterile water + sterilized acid solutions 20.00 TOTAL 100.00

The sugar content of the finished product was increased by 1.95% to compensate for the impact of acidification of the product on the organoleptic.

2. Preparation processes:

The mixture skim milk, cream, milk powder and sugar is made, pasteurized, and then fermented at 38 ° C by adding lactic ferments.

Fermentation is stopped at pH 4.50 by cooling to 4 ° C.

The white mass obtained is then supplemented with fruit preparations, acid solutions, sterile water and frozen L. plantarum according to the table presented in paragraph 3.

3. Details of the tests:

In the following table are detailed the characteristics of the different mixtures tested for 1 kilogram of finished product. <b> Table 9: </ b> Mix number 1 ' 2 ' 3 ' 4 ' Amount of lactic acid solution added (ml) 0 23.85 0 23.85 Amount of added molar phosphoric acid solution (g) 23.33 10.83 34.67 23.5 pH finished product 3.7 3.7 3.45 3.45 Lactic acidity of the finished product (g / l) 5.4 7.5 5.4 7.5 Low protonated monoacid content (g / l) 3.2 4.4 3.9 5.4 Quantity of L. plantarum added (g) 1 1 1 1 Initial population in L. plantarum (cfu / g) 1.10 ⁸ 1.10 ⁸ 1.10 ⁸ 1.10 ⁸

4. Evolution and evaluation of products during their lifetime.

In the following table are detailed the characteristics of the different mixtures tested during their aging at a storage temperature of 10 ° C. <b> Table 10: </ b> Mix number 1 ' 2 ' 3 ' 4 ' pH J0 3.7 3.7 3.45 3.45 pH D + 8 days 3.69 3.68 3.49 3.46 pH D + 22 days 3.57 3.67 3.40 3.41 pH D + 29 days 3.40 3.46 3.25 3.28 Population in initial L. plantarum (cfu / g) 1.10 ⁸ 1.10 ⁸ 1.10 ⁸ 1.10 ⁸ Population in L. plantarum at D + 22 days (cfu / g) 1,2,10 ⁸ 1.10 ⁸ 0.8.10 ⁸ 0.7.10 ⁸ Population in L. plantarum at D + 43 days (cfu / g) 1.3.10 ⁸ 0.7.10 ⁸ 0.5.10 ⁸ 0.5.10 ⁸ Visual evaluation of the swelling of the pots at D + 22 days Rating of 0 = absence at 5 = max 0 0 0 0 Evaluation of the characteristic odor of L. plantarum activity at D + 22 Note of 0 = absence at 3 = max 1 0.5 0 0 Evaluation of the characteristic taste of L. plantarum activity at D + 22 Note of 0 = absence at 3 = max 1 1 3 0

5. Conclusions:

The L. plantarum population during aging of the product is stable (ie between 0.5 × 10 ⁸ and 1.5 × 10 ⁸ cfu / g) for a pH of between 3.45 and 3.7 and with a total lactic acidity of between , 4 g / l and 7.5 g / l.

The characteristic odor of L. plantarum activity does not appear at pH 3.45 whatever the lactic acidity. On the other hand, this odor is weakly present in the products at pH 3.7 which remain very acceptable from the point of view of the consumer.

It is also noted that when the pH is lowered and the lactic acidity of the product is increased, the survival of the probiotics is reduced. Therefore, decrease the pH below 3 and increase the lactic acidity (that is to say, more generally, the content of weak monoacid whatever its form) above 7.5 g / l, induces a bacterial mortality and thus a non-stability of the probiotic.

Claims (13)

1. A method for producing a fresh food product comprising a stable concentration of live probiotics which produce false tastes and/or gas in the initial matrix of the food product, said method being characterized in that it comprises the following steps:

   a) adding 1-50 g/L of lactic acid as a dietary weak mono-acid in said initial matrix;

   b) measuring the pH of the product obtained in step a) ;

   c) adjusting the pH of the product obtained in step b) to a target pH comprised between 3 and 4, preferably comprised between 3.4 and 4, more preferably comprised between 3.4 and 3.7, by adding a stronger food acid than the dietary weak mono-acid added in step a) if the pH measured in step b) is larger than the target pH, or adding a food base if the pH measured in step b) is smaller than the target pH; and

   d) adding live probiotics to the product obtained in step c),

   so that said food product:

   contains from 1 to 20 g/L of protonated lactic acid [HA] at the end of its production as calculated by the following equation: $$\left[\mathrm{HA}\right]={\left[\mathrm{HA}\right]}_{\mathrm{Tot}}/\left(1+{10}^{\mathrm{pH}-3.86}\right)$$

   

   where [HA] represents the concentration of dietary protonated weak mono-acid
   and [HA]_Tot represents the concentration of dietary weak mono-acid, in protonated and non protonated form, has a pH comprised between 3 and 4 and
   is stored at a temperature comprised between 0 and 15°C, preferentially between 4 and 10°C.
2. The method according to claim 1, characterized in that, in step d), a concentration of live probiotics above 10⁵ CFU/mL is added, and preferably a concentration between 0.5 10⁸ and 1.5 10⁸ CFU/mL.
3. The method according to any one of claims 1 and 2, characterized in that the food acid is selected from orthophosphoric acid, citric acid, ascorbic acid, malic acid and mixtures thereof.
4. The method according to any one of claims 1 to 3, characterized in that the food base is selected from NaOH, sodium citrate, tricalcium dicitrate, phosphate buffer and citrate buffer.
5. The method according to any one of claims 1 to 4, characterized in that said food product is a drink.
6. The method according to claim 5, characterized in that said initial matrix is a vegetable juice such as fruit juice, and/or milk.
7. The method according to any one of claims 1 to 6, characterized in that said probiotics are bacterial strains.
8. The method according to claim 7, characterized in that the bacterial strains are of the Lactobacillus or Bifidobacterium genus or mixtures thereof.
9. The method according to claim 8, characterized in that said bacterial strains are Lactobacillus plantarum, preferably Lactobacillus plantarum strains deposited on 16.03.1995 under the number DSM 9843 at the Deutsche Sammlung von Mikroorganismen von Zellkulturen GmbH or Lactobacillus plantarum strains deposited on 04.04.2002 under the number CNCM I-2845 at the Collection Nationale des Cultures de Microorganismes.
10. The method according to any one of claims 7 to 9, characterized in that the concentration of bacteria in said food product is above 10⁵ CFU/mL, and preferably above 0.5 10⁸ CFU/mL.
11. The method according to any one of claims 1 to 10, characterized in that, in step a), between 5 and 50 g/L of lactic acid are added as a dietary weak mono-acid.
12. The method according to any one of claims 1 to 11, characterized in that the fresh food product contains up to 7.5 g/L of lactic acid as a dietary weak mono-acid.
13. The method according to any one of claims 1 to 12, characterized in that the target pH is between 3.4 and 3.7.